Ion channels present in outer, inner, or both membranes regulate ion flux across mitochondrial membranes. Mitochondrial ionic homeostasis regulates the membrane potential of the mitochondrion. Electron transport chain (ETC) coupling present in the inner membrane is controlled by mitochondrial membrane potential which in turn modulates ATP and reactive oxygen species (ROS) production. Mitochondrial ions also directly regulate ROS production by mitochondrial ETC complexes. Mitochondrial ions and ROS in turn regulate mitochondrial permeability transition pore (mPTP). Ionic homeostasis is coupled with mitochondrial ROS either directly or indirectly via regulating ion channels in mitochondria or other organelles. All the factors, mitochondrial ions, ROS, and ATP, modulate the cardiac function and also participate in cardioprotection against ischemia-reperfusion injury.